The invention relates to the measurement of heat changes in samples. More specifically, the invention relates to a method, a measuring cell and a system for measuring very small heat changes in a sample.
Thermodynamic data can be obtained from biological reactions by using a variety of devices. Such devices can include thermocouples, thermopiles etc.
Photoacoustic Calorimetry has been used in a wide range of different fields. Here follows some examples:
Phase transitions, Photoreaction dynamics, energetics of reactive intermediates;
Thermochemical and kinetic properties of reactive intermediates;
Information regarding inter/intra molecular reactions;
Bond dissociation enthalpies;
pKa estimations;
Key reaction parameters in important biological processes;
Radiationless decay processes in photophysical processes
Probing energetics and dynamics of fast chemical and biochemical reactions.
The list shall not be regarded as exhaustive. A number of articles about photoacoustic calometry and its different applications have been published. In an article by S. E. Braslavsky and G. E. Heibel in Chemical Review, 1992, pp. 1381-1410, the authors have carried out a review of the use of photoacoustic calorimetry for phase transitions, photoreaction dynamics and the energetics of reactive intermediates. In another article, a study was made of the thermochemical and kinetic properties of reactive intermediates of proteins using photoacoustic calometry(M. A McLean, C. Di Primo, E. Deprez, G. H. B. Hoa and S. A. Sligar, Methods in Enzymology, 295, 1998, pp. 316-330). Examples given are myoglobin and cytochrome P450cam. The authors, R. M. Borges dos Santos, A. L. C. Lagoa and J. A. M. Simoes, of an article in Journal of Chemical Thermodynamics, 31, 1999, pp. 1483-1510, describe in said article how photoacoustic calorimetry is used as a tool for high precision thermochemistry studies of transient species i.e. the reaction of phenol with di-tert-butyl peroxide. In Biophysical Journal, 79, 2000, pp. 2714-2721, an article by S. Abbruzzetti, C. Viappiani, L. J. Libertini and J. R. Small present a study of pKa measurements and the kinetics of reaction of acetate, glutamate and poly-I-glutamatic acid. The technique was applied to the quenching of the benzbphenone triplet states by dienes and the Norrish type II photoreaction of valerophenone and described by J. E. Rudski, J. L. Goodman and K. S. Peters in Journal of American Chemical Society, 107, 1985, p.7849. A paper by Kevin S. Peters and Gary J. Snyder (Science, 241, 1988, pp. 1053-1057, describes the measurement of the dynamics of enthalpy changes on the time scale of nanoseconds to microseconds for reactions initiated by the absorption of light. The method was applied to a variety of biochemical, organic and organometallic reactions.
A combined photoacoustic differential scanning calorimeter (PA-DSC) cell and an experimental set-up is known from an article by Ts. Vassilev et al.: xe2x80x9cCombined photoacoustic differential scanning calorimeter cell: Application to phase transitions.xe2x80x9d, Applied Physics A 61, (1995), pp. 129-134. The combined PA-DSC cell consists of a conventional DSC unit adapted for variable temperature studies. This is accomplished in a way that allows to perform PA and DSC measurements simultaneously and separately. FIG. 1 in the article presents the construction of the cell. It is mounted on a conventional DSC chamber at the place of the DSC cover, thus utilising the heating and cooling capabilities of the DSC instrument. The cell consists of a sample chamber and a microphone chamber. A schematic diagram of the experimental set-up is illustrated in the article""s FIG. 2.
A similar experimental set-up is also earlier known from an article by Tsvetan G. Vassilev: xe2x80x9cA combined photoacoustic DSC for simultaneous temperature modulated measurements: does it really work?xe2x80x9d, Thermochimica Acta 330 (1999), pp.145-154. The experimental setup is illustrated in the article""s FIG. 1.
The main problem in trying to obtain thermodynamic data from biological reactions is that prior art devices require the use of relatively large quantities of sample in order that meaningful results can be obtained.
It would therefore be beneficial to use a technique or device which provided some sort of signal amplification to allow the extraction of meaningful thermodynamic information from relatively small sample sizes.
The following invention describes the processes that could be used to combine the output signals derived from the microphone used in photoacoustic spectroscopy with the signal from a heat measuring device.
In short, the invention comprises a measuring cell for containing the sample during the measurement process. An electromagnetic radiation unit radiates one or several samples with modulated monochromatic or polychromatic radiation inside said measuring cell. The measuring cell comprises at least one acoustic transducer generating a first output signal and at least one heat measuring device generating a second output signal. Both output signals are connectable to a combining unit that can generate a combined output signal that can be sent to a signal processing unit.
In more detail, the present invention relates to a method according to claim 1 for measuring very small heat changes in at least one sample and determining reaction parameters. The method comprises the following steps:
modulating monochromatic or polychromatic electromagnetic radiation to excite a sample;
detecting the generated acoustic wave by the use of at least one acoustic transducer able to generate a first output signal in proportion to the heat change of the sample;
detecting a thermal wave by the use of at least one heat measuring device able to generate second output signal in proportion to the heat change of the sample;
generating at least one information signal by combining the first and the second output signals with a reference signal;
processing at least one of the information signals for determining the relevant reaction parameters.
Further, the present invention relates to a measuring cell according to claim 5 for measuring very small heat changes in at least one sample, which is/are radiated with modulated monochromatic/polychromatic electromagnetic radiation. Said measuring cell comprises at least one acoustic transducer able to generate a first output signal in proportion to the heat change of the sample, and at least one heat measuring device able to be positioned in contact with the sample, said measuring device being able to generate a second output signal in proportion to the heat change of the sample. The transducer and heat measuring device are arranged in the same main body.
Further more, the present invention relates to a system according to claim 11 for measuring very small heat changes in at least one sample and determining reaction parameters. Said system comprises a measuring cell for containing the sample during the measurement process, at least one electromagnetic radiation unit for radiating one or several samples with modulated radiation inside said measuring cell. Said measuring cell comprises at least one acoustic transducer able to generate a first output signal and at least one heat measuring device able to generate a second output signal, which signals and a reference signal are input signals to a combining unit able to generate from said signals an information signal that is connected to a signal processing unit for determining the relevant reaction parameters.
The main advantage of this combination of generated signals is the signal amplification.
The other advantage of photoacoustic spectroscopy is that it is a modulated technique. Therefore, it is very sensitive for the measurement of a small AC signal in a large DC background.